WO2022052393A1 - Method for preparing acylated anthocyanins of paeonidin and malvidin - Google Patents

Method for preparing acylated anthocyanins of paeonidin and malvidin Download PDF

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WO2022052393A1
WO2022052393A1 PCT/CN2021/070636 CN2021070636W WO2022052393A1 WO 2022052393 A1 WO2022052393 A1 WO 2022052393A1 CN 2021070636 W CN2021070636 W CN 2021070636W WO 2022052393 A1 WO2022052393 A1 WO 2022052393A1
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phase
anthocyanin
glucoside
coumaroyl
acid
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PCT/CN2021/070636
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French (fr)
Chinese (zh)
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陈卫
徐阳
谢佳宏
崔昊昕
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浙江大学
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Priority to US18/026,321 priority Critical patent/US20240025937A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C07H1/08—Separation; Purification from natural products
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D11/00—Solvent extraction
    • B01D11/02—Solvent extraction of solids
    • B01D11/0261—Solvent extraction of solids comprising vibrating mechanisms, e.g. mechanical, acoustical
    • B01D11/0265—Applying ultrasound
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D11/00—Solvent extraction
    • B01D11/02—Solvent extraction of solids
    • B01D11/0288—Applications, solvents
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D11/00—Solvent extraction
    • B01D11/04—Solvent extraction of solutions which are liquid
    • B01D11/0492—Applications, solvents used
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/16—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the fluid carrier
    • B01D15/166—Fluid composition conditioning, e.g. gradient
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/18—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns
    • B01D15/1864—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using two or more columns
    • B01D15/1871—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using two or more columns placed in series
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
    • B01D15/24—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the treatment of the fractions to be distributed
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
    • C07H17/06—Benzopyran radicals
    • C07H17/065—Benzo[b]pyrans

Definitions

  • Anthocyanins are one of the important polyphenols in grapes. Studies have shown that grapes contain delphinidin, malvain, paeoniflorin and petunidin and other anthocyanin aglycones combined with glucose to form anthocyanins. In recent years, studies have confirmed that anthocyanins derived from natural fruits and vegetables have biological activities such as antioxidant, anti-tumor, obesity control and prevention of cardiovascular disease. However, due to the sensitivity of anthocyanins to light, temperature and pH, their chemical properties are relatively unstable and their bioavailability is low, which greatly limit the practical application of anthocyanins.
  • acylated anthocyanins also have biological activity, and have more stable chemical properties and higher bioavailability. Grape contains a large amount of anthocyanin acylated on coumarin, which indicates that grape anthocyanins have better market prospects.
  • High performance liquid chromatography is a chromatography technology based on the principle of solid-liquid adsorption.
  • adsorbents such as silica gel as the stationary phase
  • separation is performed according to the difference in the binding ability of different compound molecules to the stationary phase.
  • the separation effect mainly depends on the properties of the stationary phase filler. (eg composition, particle size), and whether the LC method is good.
  • High performance liquid chromatography has the advantages of stability, reliability and good repeatability.
  • Countercurrent chromatography is a liquid-liquid chromatography technique in which both the stationary phase and the mobile phase are liquid. The principle is to separate the molecules of different compounds according to the difference in the distribution coefficients between the stationary phase and the mobile phase. The separation effect of countercurrent chromatography mainly depends on whether the two-phase solvent system used is suitable. Countercurrent chromatography has the advantages of simple sample pretreatment, wide application range, less sample loss, and large processing capacity.
  • CN104177460A discloses a method for preparing 3,5-disaccharide anthocyanins. The method uses ultrasonic-assisted extraction, extraction, purification and other steps, but the obtained product is a mixture of anthocyanins, containing three different disaccharides. Anthocyanins, and the product does not involve acylated anthocyanins.
  • CN102229633A discloses a method for separating and preparing five high-purity anthocyanin monomers from grape skins. This method uses leaching, macroporous resin purification, and liquid-phase purification to obtain five anthocyanins. However, due to the use of two-step preparation liquid-phase purification, the sample is lost and the purification yield is reduced. The purity of the anthocyanins (malvain acetylated glucoside, malvain transcoumarylated glucoside) was relatively low, only 91.7% and 95.5%, respectively.
  • CN108976268A discloses a method for preparing two main anthocyanin standard products of thorn grape.
  • the method adopts macroporous resin to adsorb and enrich the turbid juice of thorn grape, and then elute and freeze-dry to obtain crude anthocyanins.
  • Countercurrent chromatography using water-n-butanol-methyl tert-butyl ether-acetonitrile-trifluoroacetic acid (volume ratio 5:4:1:2:0.001 or 5:3:1:1:0.001) as a two-phase solvent system After separation, two anthocyanins were obtained with purities of 95.8% and 92.2%, respectively.
  • the present invention provides a method for separating and preparing paeoniflorin-3-O-(6-p-coumaroyl) glucoside and malvain-3-O-(6-p-coumaroyl) ) method for glucoside, according to the characteristics of acylated anthocyanins, the present invention combines preparation liquid chromatography with high-speed countercurrent chromatography, which can realize the preparation of high-purity paeoniflorin-3- O-(6-p-coumaroyl) glucoside and malvain-3-O-(6-p-coumaroyl) glucoside provide new ideas for the development and utilization of grape resources in my country.
  • a method for separating and preparing paeoniflorin-3-O-(6-p-coumaroyl) glucoside and malvain-3-O-(6-p-coumaroyl) glucoside comprising the steps of:
  • phase A is pure acetonitrile
  • phase B is formic acid aqueous solution with a volume percent concentration of formic acid of 1% to 2%;
  • the gradient elution program is: 0-4min, 5%-20% phase A; 4-18min, 20%-25% phase A; 18-21min, 25%-35% phase A; 21-24min, 35%-60 %A phase; 24-27min, 60%-5%A phase; 27-30min, 5%A phase;
  • the flow rate is 8-10mL/min, the column temperature is 30°C, and the detection wavelength is 520nm;
  • high-speed countercurrent chromatographic separation mix ethyl acetate, water and trifluoroacetic acid in a volume ratio of 1:1:0.001 as a two-phase solvent system, and the upper phase is a stationary phase, and the lower phase is a mobile phase, and the stationary phase is sequentially
  • the phase and mobile phase are pumped into the high-speed countercurrent chromatographic instrument. After the two phases reach equilibrium in the pipeline, the crude anthocyanin monomer is dissolved in the mobile phase, and the sample is injected and detected under an ultraviolet detector.
  • the detection wavelength is 280nm, and the collected The components with retention time of 116-126min and 90-100min are concentrated under reduced pressure and freeze-dried to obtain paeoniflorin-3-O-(6-p-coumaroyl)glucoside and malvain-3-O-( 6-p-coumaroyl) glucoside.
  • the percentages of raw materials in the present invention all refer to the volume percentage concentration, and the various solutions in the present invention, unless otherwise specified, use water as the solvent.
  • step (1) the acidic alcohol solution is extracted and concentrated, specifically: washing the grapes and taking the skins, mixing with the acidic alcohol solution, beating after mixing, ultrasonically extracting below 50°C (preferably at room temperature), filtering, and the filtrate is Concentrate under reduced pressure at 40-50°C to remove ethanol to obtain a crude extract of grape skin anthocyanins;
  • the material-to-liquid ratio of grape skin and acidic ethanol solution is 1g:4-8mL;
  • the volume concentration of ethanol is 50%-80%, preferably 60%-70%, and the volume concentration of acid is 0.1%-1%;
  • the ultrasonic extraction time is 40-120min.
  • the acid is selected from at least one of hydrochloric acid, formic acid, acetic acid, and oxalic acid.
  • step (2) the macroporous resin purification method is specifically:
  • the crude extract of grape skin anthocyanins was injected into the macroporous resin, and then eluted with 0, 5%, 20%, 40%, and 60% acidic ethanol solutions of 4 times the column volume (4BV) in sequence, and collected.
  • the acidic ethanol eluent with ethanol volume concentration of 40% and 60% is evaporated under reduced pressure at 40-50°C to remove the ethanol to obtain the anthocyanin eluent;
  • the macroporous resin is preferably selected from AB-8, D101, XAD-7, HPD-100 or DM-130, its specific surface area is 450-550m2/g, the average pore size is 10-50nm, and the particle size is in the range of 0.3-1.25mm ;
  • the acidic ethanol solution is selected from an ethanol solution with an acid volume percentage concentration of 0.1% to 1.5%, wherein the acid is selected from at least one of hydrochloric acid, formic acid, acetic acid, and oxalic acid.
  • step (3) the organic solvent is ethyl acetate.
  • step (3) it is preferable to extract at a ratio of the organic solvent to the anthocyanin eluent volume ratio of 1:1, and extract more than 2 times.
  • step (4) the lyophilized anthocyanin powder can be dissolved in phase B or water.
  • the liquid chromatographic column used by the preparative liquid chromatography system is a C18 column, and the single injection volume is 10-40 mg in terms of anthocyanin freeze-dried powder, and the volume after the evaporation under reduced pressure is before the evaporation. 40%-70% of the volume.
  • step (5) the temperature of the high-speed countercurrent chromatographic instrument is stabilized at 20-30 ° C, the positive connection is forward, and the stationary phase is pumped, and then the rotating speed is adjusted to 800-950 r/min, and the mobile phase is passed into the mobile phase at a flow rate of 2 mL/min. Balanced, the amount of each injection is 20-50 mg based on crude anthocyanin monomer.
  • the main advantages of the present invention include:
  • the simultaneous separation of paeoniflorin-3-O-(6-p-coumaroyl) glucoside (molecular structure shown in Figure 1) and malvain-3-O-(6-p
  • the method for coumaroyl) glucoside (molecular structure is shown in Figure 2), the yield of paeoniflorin-3-O-(6-p-coumaroyl) glucoside can be not less than 6mg/kg grape skin, malvain
  • the yield of -3-O-(6-p-coumaroyl) glucoside monomer can be no less than 20 mg/kg grape skin, and the purity of both products is no less than 98%.
  • paeoniflorin-3-O-(6-p-coumaroyl) glucoside and Malvain-3-O-(6-p-coumaroyl) glucoside has the advantages of large processing capacity and good repeatability, and is convenient for industrialized production.
  • Fig. 1 is the molecular structure diagram of paeoniflorin-3-O-(6-p-coumaroyl) glucoside;
  • Fig. 2 is the molecular structure diagram of malvain-3-O-(6-p-coumaroyl) glucoside
  • Fig. 3 is in embodiment 1, the high performance liquid chromatogram of grape skin anthocyanin crude extract
  • Fig. 4 is in Example 1, after separation and purification by macroporous resin, containing paeoniflorin-3-O-(6-p-coumaroyl) glucoside, malvain-3-O-(6-p-coumaroyl) ) the high performance liquid chromatogram of the eluent of two anthocyanins of glucoside;
  • Fig. 5 is the high-speed countercurrent chromatogram in embodiment 1;
  • Fig. 6 is in embodiment 1, the high performance liquid chromatogram of final product Paeoniflorin-3-O-(6-p-coumaroyl) glucoside monomer;
  • Fig. 7 is in embodiment 1, the high performance liquid chromatogram of final product malvain-3-O-(6-p-coumaroyl) glucoside monomer;
  • Fig. 8 is the secondary mass spectrum of paeoniflorin-3-O-(6-p-coumaroyl) glucoside and malvain-3-O-(6-p-coumaroyl) glucoside;
  • FIG. 9 is a high-performance liquid chromatogram of the final product of Comparative Example 2.
  • the AB-8 macroporous resin was loaded into the chromatographic column, and ethanol, 0.5mol/L hydrochloric acid solution, 0.5mol/L sodium hydroxide solution were successively used, and after washing with water, the anthocyanin crude extract was subjected to 0.2BV/L.
  • the flow rate of h is injected into the chromatography column.
  • acid water containing 0.5% hydrochloric acid
  • 5%, 20%, 40%, and 60% acidic ethanol containing 0.5% hydrochloric acid
  • the ethanol eluent was evaporated under reduced pressure to remove ethanol.
  • Liquid-phase separation was performed using an Ultimate XB-C18 (7 ⁇ m, 21.2 ⁇ 250 mm) preparative chromatographic column.
  • the mobile phase consisted of pure acetonitrile (phase A) and 1.5% formic acid in water (phase B).
  • the gradient elution method is as follows: 0-4min, 5%-20% phase A; 4-18min, 20%-25% phase A; 18-21min, 25%-35% phase A; 21-24min, 35%-60 %A phase; 24-27min, 60%-5%A phase; 27-30min, 5%A phase, flow rate is 10mL/min, column temperature is 30°C, detection wavelength is 520nm.
  • the lyophilized anthocyanin powder was dissolved in phase B and then injected, and the injection volume was 4 mL.
  • the fractions of 22.0-23.5 min were collected, concentrated under appropriate reduced pressure, and then freeze-dried to obtain paeoniflorin-3-O-(6-paraben Crude monomers of myoyl) glucoside and malvain-3-O-(6-p-coumaroyl) glucoside.
  • Ethyl acetate, water and trifluoroacetic acid were placed in a separatory funnel in a volume ratio of 1:1:0.001, shaken well, and after standing for 30 min, the upper and lower phases were separated, and ultrasonically degassed for 30 min respectively.
  • the crude lyophilized powder of anthocyanin monomer was dissolved in 1 mL of mobile phase, filtered with a microporous membrane, and injected into the sample.
  • a single injection of 10 mL was performed and detected under an ultraviolet detector with a detection wavelength of 280 nm.
  • the components of 90-100min and 116-126min were collected respectively (as shown in Figure 5) and concentrated under reduced pressure, and lyophilized to obtain 6.8mg of Paeoniflorin-3-O-(6-p-coumaroyl) glucoside.
  • the chromatogram is shown in Figure 6, the HPLC purity is 99.3%, and 24 mg of malvain-3-O-(6-p-coumaroyl) glucoside, the high performance liquid chromatogram is shown in Figure 7, and the HPLC purity is 98.7%.
  • the prepared anthocyanin sample was injected into the mass spectrometer, and the sample was analyzed according to the mass spectrum (Fig. 8), and it was confirmed that the mass number of the separated anthocyanin was normal.
  • the grapes were washed and peeled to obtain 2kg of grape skins, and 80% ethanol solution containing 0.5% (v/v) hydrochloric acid was added according to the ratio of material to liquid ratio of 1g: 6mL to fully mix, and ultrasonically extracted for 60min, (control temperature below 50°C) , protected from light), filtered with gauze, the filtrate was centrifuged at 4000 rpm for 10 min, and the supernatant was taken. The filter residue was extracted once more in the same way. The filtrates were combined and filtered again using a Buchner funnel. The filtrate was evaporated under reduced pressure at 45°C to remove ethanol and concentrated to obtain a crude extract of grape skin anthocyanins.
  • the AB-8 macroporous resin was loaded into the chromatographic column, and ethanol, 0.5mol/L hydrochloric acid solution, 0.5mol/L sodium hydroxide solution were successively used, and after washing with water, the anthocyanin crude extract was subjected to 0.2BV/L.
  • the flow rate of h is injected into the chromatography column.
  • use acid water (containing 0.5% hydrochloric acid), 5%, 20%, 40%, and 60% acidic ethanol (containing 0.5% hydrochloric acid) to elute with 4 column volumes each, and collect 40% and 60% acid
  • the ethanol eluent was evaporated under reduced pressure to remove ethanol.
  • the mixture was extracted three times with ethyl acetate, and the aqueous phase was taken, concentrated under appropriate reduced pressure, and freeze-dried to obtain freeze-dried anthocyanin powder.
  • Liquid-phase separation was performed using an Ultimate XB-C18 (7 ⁇ m, 21.2 ⁇ 250 mm) preparative chromatographic column.
  • the mobile phase consisted of pure acetonitrile (phase A) and 1.5% formic acid in water (phase B).
  • the gradient elution method is as follows: 0-4min, 5%-20% phase A; 4-18min, 20%-25% phase A; 18-21min, 25%-35% phase A; 21-24min, 35%-60 %A phase; 24-27min, 60%-5%A phase; 27-30min, 5%A phase, flow rate is 10mL/min, column temperature is 30°C, detection wavelength is 520nm.
  • the lyophilized anthocyanin powder was dissolved in phase B and then injected, and the injection volume was 4 mL.
  • the fractions of 22.0-23.5 min were collected, concentrated under appropriate reduced pressure, and then freeze-dried to obtain paeoniflorin-3-O-(6-paraben Crude monomers of myoyl) glucoside and malvain-3-O-(6-p-coumaroyl) glucoside.
  • Ethyl acetate, water and trifluoroacetic acid were placed in a separatory funnel in a volume ratio of 1:1:0.001, shaken well, and after standing for 30 min, the upper and lower phases were separated, and ultrasonically degassed for 30 min respectively.
  • the crude lyophilized powder of anthocyanin monomer was dissolved in 1 mL of mobile phase, filtered with a microporous membrane, and injected into the sample.
  • the AB-8 macroporous resin was loaded into the chromatographic column, and ethanol, 0.5mol/L hydrochloric acid solution, 0.5mol/L sodium hydroxide solution were successively used, and after washing with water, the anthocyanin crude extract was subjected to 0.2BV/L.
  • the flow rate of h is injected into the chromatography column.
  • use acid water (containing 0.5% hydrochloric acid), 5%, 20%, 40%, and 60% acidic ethanol (containing 0.5% hydrochloric acid) to elute with 4 column volumes each, and collect 40% and 60% acid
  • the ethanol eluent was evaporated under reduced pressure to remove ethanol.
  • the mixture was extracted twice with ethyl acetate, and the aqueous phase was taken, concentrated under appropriate reduced pressure, and lyophilized to obtain lyophilized anthocyanin powder.
  • the lyophilized anthocyanin powder was dissolved in phase B and then injected, and the injection volume was 4 mL.
  • the fractions of 22.0-23.5 min were collected, concentrated under appropriate reduced pressure, and then freeze-dried to obtain paeoniflorin-3-O-(6-paraben Crude monomers of myoyl) glucoside and malvain-3-O-(6-p-coumaroyl) glucoside.
  • Ethyl acetate, water and trifluoroacetic acid were placed in a separatory funnel in a volume ratio of 1:1:0.001, shaken well, and after standing for 30 min, the upper and lower phases were separated, and ultrasonically degassed for 30 min respectively.
  • the instrument temperature of the high-speed countercurrent chromatography system was stabilized at 20 °C, the stationary phase was pumped, and then the rotational speed was adjusted to 850 r/min, the positive rotation was connected, and the mobile phase was passed into the mobile phase at a flow rate of 2 mL/min until equilibrium.
  • the fractions of 90-100min and 116-126min were collected and concentrated under reduced pressure, and lyophilized to obtain 62 mg of Paeoniflorin-3-O-(6-p-coumaroyl) glucoside, the HPLC purity was 98.5%, and 210 mg of Malvain-3-O-(6-p-coumaroyl)glucoside, HPLC purity 98.2%.
  • the preparation process is the same as that of Example 1, except that the extraction process is changed not to use acid ethanol solution extraction, but to use acid-free ethanol solution extraction instead.
  • the other steps remain unchanged, the yield of the final target product, paeoniflorin-3-O-(6-p-coumaroyl) glucoside, is 2 mg/kg grape skin, which is much lower than 6 mg/kg grape skin; malvain-3
  • the yield of -O-(6-p-coumaroyl) glucoside monomer was 7 mg/kg grape skin, which was much lower than 20 mg/kg grape skin.
  • the preparation process is the same as that of Example 1, the difference is only that the step of high-speed countercurrent chromatography purification is removed, and other steps remain unchanged, and the final product obtained can only obtain paeoniflorin-3-O-(6-p-coumaroyl) glucoside and bromine.
  • the preparation process is the same as that of Example 1, except that the solvent system separated by high-speed countercurrent chromatography is replaced with water-n-butanol-methyl tert-butyl ether-acetonitrile-trifluoroacetic acid in a volume ratio of 5:4:1:2 : system of 0.001.
  • the target compounds paeoniflorin-3-O-(6-p-coumaroyl) glucoside and malvain-3-O-(6-p-coumaroyl) glucoside were mainly retained in the upper phase and could not be obtained.
  • the preparation process is the same as that of Example 1, except that the solvent system separated by high-speed countercurrent chromatography is replaced with a system with a volume ratio of ethyl acetate-water-trifluoroacetic acid of 1:2:0.001.
  • the target compounds paeoniflorin-3-O-(6-p-coumaroyl) glucoside and malvain-3-O-(6-p-coumaroyl) glucoside could not be obtained.
  • the preparation process is the same as that of Example 1, except that the solvent system separated by high-speed countercurrent chromatography is replaced with a system with a volume ratio of ethyl acetate-water-trifluoroacetic acid of 2:1:0.001.
  • the target compounds paeoniflorin-3-O-(6-p-coumaroyl) glucoside and malvain-3-O-(6-p-coumaroyl) glucoside could not be obtained.

Abstract

A method for separating and preparing paeonidin-3-O-(6-p-coumaroyl) glucoside and malvidin-3-O-(6-p-coumaroyl) glucoside. Two high-purity acylated paeonidin and malvidin anthocyanin monomers are purified and separated from grapes by means of steps of abstraction, macroporous resin purification, extraction, preparative liquid phase chromatography and high speed countercurrent chromatography, etc. By means of said method, at least 60 mg of paeonidin-3-O-(6-p-coumaroyl) glucoside and at least 200 mg of malvidin-3-O-(6-p-coumaroyl) glucoside can be obtained from 10 kg of grape skin, and each purity can reach 98% or more. The present method has the advantages of simple operation, large processing volume, good repeatability, etc. and provides a new idea for the development and utilization of grape resources.

Description

一种芍药素和锦葵素酰基化花色苷的制备方法A kind of preparation method of paeoniflorin and malvain acylated anthocyanin 技术领域technical field
本发明涉及天然产物的分离纯化技术领域，具体涉及一种分离制备芍药素-3-O-(6-对香豆酰)葡萄糖苷和锦葵素-3-O-(6-对香豆酰)葡萄糖苷的方法。The invention relates to the technical field of separation and purification of natural products, in particular to a kind of separation and preparation of paeoniflorin-3-O-(6-p-coumaroyl) glucoside and malvain-3-O-(6-p-coumaroyl) ) glucoside method.
背景技术Background technique
花色苷是葡萄中重要的多酚类物质之一，研究表明，葡萄中含有飞燕草素、锦葵素、芍药素和矮牵牛素等花青素苷元与葡萄糖结合形成的花色苷。近年来的研究证实了天然果蔬来源的花色苷具有抗氧化、抗肿瘤、控制肥胖和预防心血管疾病等生物活性。但由于花色苷对光、温度和pH敏感，导致其化学性质相对不稳定，而且生物利用度较低，这些性质极大地限制了花色苷的实际应用。已有研究表明，相对于非酰化花色苷，酰化花色苷同样具有生物活性，并且有更稳定的化学性质以及更高的生物利用度。而葡萄中含有大量对香豆素酰化的花色苷，这说明葡萄花色苷具有更好的市场前景。Anthocyanins are one of the important polyphenols in grapes. Studies have shown that grapes contain delphinidin, malvain, paeoniflorin and petunidin and other anthocyanin aglycones combined with glucose to form anthocyanins. In recent years, studies have confirmed that anthocyanins derived from natural fruits and vegetables have biological activities such as antioxidant, anti-tumor, obesity control and prevention of cardiovascular disease. However, due to the sensitivity of anthocyanins to light, temperature and pH, their chemical properties are relatively unstable and their bioavailability is low, which greatly limit the practical application of anthocyanins. Studies have shown that, compared with non-acylated anthocyanins, acylated anthocyanins also have biological activity, and have more stable chemical properties and higher bioavailability. Grape contains a large amount of anthocyanin acylated on coumarin, which indicates that grape anthocyanins have better market prospects.
近年来，固相萃取技术(SPE)、制备型高效液相色谱技术(preparative-HPLC)和高速逆流色谱技术(HSCCC)等新型纯化技术开始得到发展和应用。高效液相色谱是基于固液吸附原理的色谱技术，通过使用硅胶等吸附剂作为固定相，根据不同化合物分子与固定相的结合能力的差异性而分离，分离效果主要取决于固定相填料的性质(如组成，粒径大小)，以及液相色谱方法是否良好。高效液相色谱技术具有稳定、可靠、重复性好等优点。逆流色谱是一种液液色谱技术，其固定相和流动相都是液态的，原理是根据不同化合物分子在固定相和流动相的分配系数的差异而分离。逆流色谱法的分离效果，主要取决于使用的两相溶剂体系是否合适。逆流色谱技术具有样品前处理简单、适用范围广、样品损失少、处理量大等优点。In recent years, new purification technologies such as solid phase extraction (SPE), preparative high performance liquid chromatography (preparative-HPLC) and high-speed countercurrent chromatography (HSCCC) have begun to be developed and applied. High performance liquid chromatography is a chromatography technology based on the principle of solid-liquid adsorption. By using adsorbents such as silica gel as the stationary phase, separation is performed according to the difference in the binding ability of different compound molecules to the stationary phase. The separation effect mainly depends on the properties of the stationary phase filler. (eg composition, particle size), and whether the LC method is good. High performance liquid chromatography has the advantages of stability, reliability and good repeatability. Countercurrent chromatography is a liquid-liquid chromatography technique in which both the stationary phase and the mobile phase are liquid. The principle is to separate the molecules of different compounds according to the difference in the distribution coefficients between the stationary phase and the mobile phase. The separation effect of countercurrent chromatography mainly depends on whether the two-phase solvent system used is suitable. Countercurrent chromatography has the advantages of simple sample pretreatment, wide application range, less sample loss, and large processing capacity.
目前在葡萄花色苷的制备中，主要采用萃取、大孔树脂和单一的柱层析或色谱技术进行分离纯化。如CN104177460A中公开了一种3,5-二糖类 花色苷的制备方法，该方法使用超声辅助提取、萃取、纯化等步骤，但获得的产物为花色苷混合物，含有三种不同的二糖类花色苷，并且产物不涉及酰化花色苷。At present, in the preparation of grape anthocyanins, extraction, macroporous resin and single column chromatography or chromatographic technology are mainly used for separation and purification. For example, CN104177460A discloses a method for preparing 3,5-disaccharide anthocyanins. The method uses ultrasonic-assisted extraction, extraction, purification and other steps, but the obtained product is a mixture of anthocyanins, containing three different disaccharides. Anthocyanins, and the product does not involve acylated anthocyanins.
又如CN102229633A中公开了一种从葡萄皮中分离制备五种高纯度花色苷单体的方法。该方法使用浸提，大孔树脂纯化，制备液相的方法纯化得到了五种花色苷，然而由于使用了两步制备液相纯化，导致样品损耗，降低了纯化得率，且其中两种酰化花色苷(锦葵素乙酰化葡萄糖苷，锦葵素反式香豆酰化葡萄糖苷)的纯度相对较低，分别仅为91.7％和95.5％。Another example is CN102229633A, which discloses a method for separating and preparing five high-purity anthocyanin monomers from grape skins. This method uses leaching, macroporous resin purification, and liquid-phase purification to obtain five anthocyanins. However, due to the use of two-step preparation liquid-phase purification, the sample is lost and the purification yield is reduced. The purity of the anthocyanins (malvain acetylated glucoside, malvain transcoumarylated glucoside) was relatively low, only 91.7% and 95.5%, respectively.
CN108976268A中公开了一种制备刺葡萄两个主要花色苷标准品的方法，该方法采用大孔树脂对刺葡萄浊汁进行吸附富集，随后经过洗脱，冷冻干燥得到花色苷粗品，再使用高速逆流色谱，以水-正丁醇-甲基叔丁基醚-乙腈-三氟乙酸(体积比5：4：1：2：0.001或5：3：1：1：0.001)为两相溶剂体系分离，得到两种花色苷，纯度分别为95.8％和92.2％。但是从刺葡萄浊汁HPLC图中，可以看到其花色苷组成简单，由高速逆流色谱的局限性可推测出，当分离的样品花色苷组分复杂时，使用该方法可能难以得到目标花色苷。CN108976268A discloses a method for preparing two main anthocyanin standard products of thorn grape. The method adopts macroporous resin to adsorb and enrich the turbid juice of thorn grape, and then elute and freeze-dry to obtain crude anthocyanins. Countercurrent chromatography, using water-n-butanol-methyl tert-butyl ether-acetonitrile-trifluoroacetic acid (volume ratio 5:4:1:2:0.001 or 5:3:1:1:0.001) as a two-phase solvent system After separation, two anthocyanins were obtained with purities of 95.8% and 92.2%, respectively. However, from the HPLC chart of the turbid grape juice, it can be seen that the composition of anthocyanins is simple. From the limitations of high-speed countercurrent chromatography, it can be inferred that when the anthocyanin components of the separated sample are complex, it may be difficult to obtain the target anthocyanins using this method. .
由于酰化花色苷的分离纯化困难，导致目前市面上没有商业化的酰化花色苷标准品。因此，研究开发一种从复杂花色苷原料如葡萄中纯化酰化花色苷单体的工艺，对推动花色苷标准品市场及开发葡萄深加工产品具有重要意义。Due to the difficulty in the separation and purification of acylated anthocyanins, there is currently no commercial acylated anthocyanin standard on the market. Therefore, research and development of a process for purifying acylated anthocyanin monomers from complex anthocyanin raw materials such as grapes is of great significance for promoting the market of anthocyanin standard products and developing grape deep-processed products.
发明内容SUMMARY OF THE INVENTION
针对本领域存在的不足之处，本发明提供了一种分离制备芍药素-3-O-(6-对香豆酰)葡萄糖苷和锦葵素-3-O-(6-对香豆酰)葡萄糖苷的方法，本发明根据酰化花色苷的特性，将制备液相色谱与高速逆流色谱联用，可以实现从花色苷组分复杂的葡萄中，大量制备高纯度的芍药素-3-O-(6-对香豆酰)葡萄糖苷和锦葵素-3-O-(6-对香豆酰)葡萄糖苷，为我国葡萄资源的开发利用提供新的思路。In view of the deficiencies in the art, the present invention provides a method for separating and preparing paeoniflorin-3-O-(6-p-coumaroyl) glucoside and malvain-3-O-(6-p-coumaroyl) ) method for glucoside, according to the characteristics of acylated anthocyanins, the present invention combines preparation liquid chromatography with high-speed countercurrent chromatography, which can realize the preparation of high-purity paeoniflorin-3- O-(6-p-coumaroyl) glucoside and malvain-3-O-(6-p-coumaroyl) glucoside provide new ideas for the development and utilization of grape resources in my country.
一种分离制备芍药素-3-O-(6-对香豆酰)葡萄糖苷和锦葵素-3-O-(6-对香豆酰)葡萄糖苷的方法，包括步骤：A method for separating and preparing paeoniflorin-3-O-(6-p-coumaroyl) glucoside and malvain-3-O-(6-p-coumaroyl) glucoside, comprising the steps of:
(1)醇提浓缩：以葡萄为原料，经酸性醇溶液提取，浓缩得到葡萄 皮花色苷粗提液；(1) alcohol extraction and concentration: take grape as raw material, extract through acid alcohol solution, concentrate to obtain grape skin anthocyanin crude extract;
(2)大孔树脂纯化：将所述葡萄皮花色苷粗提液注入大孔树脂，经洗脱，浓缩得到花色苷洗脱液；(2) macroporous resin purification: the crude extract of grape skin anthocyanins is injected into macroporous resin, elution, and concentration to obtain anthocyanin eluent;
(3)萃取：使用有机溶剂萃取所述花色苷洗脱液，再经减压浓缩，冻干后得到花色苷冻干粉；(3) extraction: use organic solvent to extract described anthocyanin eluent, then concentrate under reduced pressure, obtain anthocyanin freeze-dried powder after freeze-drying;
(4)制备液相色谱纯化：将所述花色苷冻干粉溶解后，注入制备型液相色谱系统中，并用紫外检测器下检测，具体参数条件如下：(4) purification by preparative liquid chromatography: after dissolving the anthocyanin freeze-dried powder, inject it into a preparative liquid chromatography system, and detect with an ultraviolet detector, and the specific parameter conditions are as follows:
流动相：A相为纯乙腈，B相为甲酸体积百分浓度为1％～2％的甲酸水溶液；Mobile phase: phase A is pure acetonitrile, phase B is formic acid aqueous solution with a volume percent concentration of formic acid of 1% to 2%;
梯度洗脱程序为：0-4min，5％-20％A相；4-18min，20％-25％A相；18-21min，25％-35％A相；21-24min，35％-60％A相；24-27min，60％-5％A相；27-30min，5％A相；The gradient elution program is: 0-4min, 5%-20% phase A; 4-18min, 20%-25% phase A; 18-21min, 25%-35% phase A; 21-24min, 35%-60 %A phase; 24-27min, 60%-5%A phase; 27-30min, 5%A phase;
流速为8-10mL/min，柱温为30℃，检测波长为520nm；The flow rate is 8-10mL/min, the column temperature is 30℃, and the detection wavelength is 520nm;
根据液相色谱图收集保留时间为22.0-23.5min的组分，之后减压蒸发，冻干，得到花色苷单体粗品；The components with retention time of 22.0-23.5min were collected according to the liquid chromatogram, then evaporated under reduced pressure and freeze-dried to obtain the crude anthocyanin monomer;
(5)高速逆流色谱分离：将乙酸乙酯、水和三氟乙酸以1:1:0.001的体积比混合作为两相溶剂体系，且以上相为固定相，下相为流动相，依次将固定相和流动相泵入高速逆流色谱仪器中，两相在管路中达到平衡后，将花色苷单体粗品用流动相溶解，进样并在紫外检测器下检测，检测波长为280nm，分别收集保留时间为116-126min和90-100min的组分并减压浓缩、冻干，分别得到芍药素-3-O-(6-对香豆酰)葡萄糖苷和锦葵素-3-O-(6-对香豆酰)葡萄糖苷。(5) high-speed countercurrent chromatographic separation: mix ethyl acetate, water and trifluoroacetic acid in a volume ratio of 1:1:0.001 as a two-phase solvent system, and the upper phase is a stationary phase, and the lower phase is a mobile phase, and the stationary phase is sequentially The phase and mobile phase are pumped into the high-speed countercurrent chromatographic instrument. After the two phases reach equilibrium in the pipeline, the crude anthocyanin monomer is dissolved in the mobile phase, and the sample is injected and detected under an ultraviolet detector. The detection wavelength is 280nm, and the collected The components with retention time of 116-126min and 90-100min are concentrated under reduced pressure and freeze-dried to obtain paeoniflorin-3-O-(6-p-coumaroyl)glucoside and malvain-3-O-( 6-p-coumaroyl) glucoside.
如无特殊说明，本发明中出现的原料百分比均指体积百分浓度，本发明中出现的各种溶液，如无特别说明，均以水作为溶剂。Unless otherwise specified, the percentages of raw materials in the present invention all refer to the volume percentage concentration, and the various solutions in the present invention, unless otherwise specified, use water as the solvent.
步骤(1)中，所述酸性醇溶液提取，浓缩，具体为：将葡萄洗净取皮，与酸性乙醇溶液混合，混匀后打浆，50℃以下(优选室温)超声提取，过滤，滤液在40-50℃减压浓缩除去乙醇，得到葡萄皮花色苷粗提液；In step (1), the acidic alcohol solution is extracted and concentrated, specifically: washing the grapes and taking the skins, mixing with the acidic alcohol solution, beating after mixing, ultrasonically extracting below 50°C (preferably at room temperature), filtering, and the filtrate is Concentrate under reduced pressure at 40-50°C to remove ethanol to obtain a crude extract of grape skin anthocyanins;
葡萄皮与酸性乙醇溶液的料液比为1g:4-8mL；The material-to-liquid ratio of grape skin and acidic ethanol solution is 1g:4-8mL;
所述酸性乙醇溶液中，乙醇体积浓度为50％-80％，优选60％-70％，酸体积浓度为0.1％-1％；In the acidic ethanol solution, the volume concentration of ethanol is 50%-80%, preferably 60%-70%, and the volume concentration of acid is 0.1%-1%;
所述超声提取时间为40-120min。The ultrasonic extraction time is 40-120min.
步骤(1)中，所述酸性乙醇溶液中，酸选自盐酸、甲酸、乙酸、草酸中的至少一种。In step (1), in the acidic ethanol solution, the acid is selected from at least one of hydrochloric acid, formic acid, acetic acid, and oxalic acid.
步骤(2)中，所述大孔树脂纯化方法具体为：In step (2), the macroporous resin purification method is specifically:
将葡萄皮花色苷粗提液注入大孔树脂中，之后依次用乙醇体积浓度为0，5％，20％，40％，60％的酸性乙醇溶液各4倍柱体积(4BV)洗脱，收集乙醇体积浓度为40％和60％的酸性乙醇洗脱液，40-50℃减压蒸发除去乙醇，得到花色苷洗脱液；The crude extract of grape skin anthocyanins was injected into the macroporous resin, and then eluted with 0, 5%, 20%, 40%, and 60% acidic ethanol solutions of 4 times the column volume (4BV) in sequence, and collected. The acidic ethanol eluent with ethanol volume concentration of 40% and 60% is evaporated under reduced pressure at 40-50°C to remove the ethanol to obtain the anthocyanin eluent;
所述大孔树脂优选自AB-8，D101，XAD-7，HPD-100或DM-130，其比表面积为450-550m2/g，平均孔径为10-50nm，粒径范围在0.3-1.25mm；The macroporous resin is preferably selected from AB-8, D101, XAD-7, HPD-100 or DM-130, its specific surface area is 450-550m2/g, the average pore size is 10-50nm, and the particle size is in the range of 0.3-1.25mm ;
步骤(2)中，所述酸性乙醇溶液选自酸的体积百分浓度为0.1％～1.5％的乙醇溶液，其中酸选自盐酸、甲酸、乙酸、草酸中的至少一种。In step (2), the acidic ethanol solution is selected from an ethanol solution with an acid volume percentage concentration of 0.1% to 1.5%, wherein the acid is selected from at least one of hydrochloric acid, formic acid, acetic acid, and oxalic acid.
步骤(3)中，所述有机溶剂为乙酸乙酯。In step (3), the organic solvent is ethyl acetate.
步骤(3)中，优选以所述有机溶剂与花色苷洗脱液体积比1:1的比例萃取，并萃取2次以上。In step (3), it is preferable to extract at a ratio of the organic solvent to the anthocyanin eluent volume ratio of 1:1, and extract more than 2 times.
步骤(4)中，所述花色苷冻干粉可以用B相或水溶解。In step (4), the lyophilized anthocyanin powder can be dissolved in phase B or water.
步骤(4)中，制备型液相色谱系统使用的液相色谱柱为C18柱，单次进样量以花色苷冻干粉计为10-40mg，所述减压蒸发后的体积为蒸发前体积的40％-70％。In step (4), the liquid chromatographic column used by the preparative liquid chromatography system is a C18 column, and the single injection volume is 10-40 mg in terms of anthocyanin freeze-dried powder, and the volume after the evaporation under reduced pressure is before the evaporation. 40%-70% of the volume.
步骤(5)中，高速逆流色谱仪器温度稳定在20-30℃，正接正转，泵入固定相，之后调节转速至800-950r/min，以2mL/min的流速通入流动相并使之平衡，每次进样量以花色苷单体粗品计为20-50mg。In step (5), the temperature of the high-speed countercurrent chromatographic instrument is stabilized at 20-30 ° C, the positive connection is forward, and the stationary phase is pumped, and then the rotating speed is adjusted to 800-950 r/min, and the mobile phase is passed into the mobile phase at a flow rate of 2 mL/min. Balanced, the amount of each injection is 20-50 mg based on crude anthocyanin monomer.
本发明与现有技术相比，主要优点包括：Compared with the prior art, the main advantages of the present invention include:
1、首次建立了从葡萄皮中同时分离芍药素-3-O-(6-对香豆酰)葡萄糖苷(分子结构如图1所示)和锦葵素-3-O-(6-对香豆酰)葡萄糖苷(分子结构如图2所示)的方法，芍药素-3-O-(6-对香豆酰)葡萄糖苷的产率可不低于6mg/kg葡萄皮，锦葵素-3-O-(6-对香豆酰)葡萄糖苷单体的产率可不低于20mg/kg葡萄皮，两种产物的纯度均不低于98％。1. For the first time, the simultaneous separation of paeoniflorin-3-O-(6-p-coumaroyl) glucoside (molecular structure shown in Figure 1) and malvain-3-O-(6-p The method for coumaroyl) glucoside (molecular structure is shown in Figure 2), the yield of paeoniflorin-3-O-(6-p-coumaroyl) glucoside can be not less than 6mg/kg grape skin, malvain The yield of -3-O-(6-p-coumaroyl) glucoside monomer can be no less than 20 mg/kg grape skin, and the purity of both products is no less than 98%.
2、通过将制备液相色谱和高速逆流色谱联用，能够在多酚组分复杂的葡萄原料中，大批量地制备得到芍药素-3-O-(6-对香豆酰)葡萄糖苷和锦葵素-3-O-(6-对香豆酰)葡萄糖苷，有处理量大、重复性好等优点，便于实 现工业化生产。2. By combining preparative liquid chromatography and high-speed countercurrent chromatography, paeoniflorin-3-O-(6-p-coumaroyl) glucoside and Malvain-3-O-(6-p-coumaroyl) glucoside has the advantages of large processing capacity and good repeatability, and is convenient for industrialized production.
附图说明Description of drawings
图1为芍药素-3-O-(6-对香豆酰)葡萄糖苷的分子结构图；Fig. 1 is the molecular structure diagram of paeoniflorin-3-O-(6-p-coumaroyl) glucoside;
图2为锦葵素-3-O-(6-对香豆酰)葡萄糖苷的分子结构图；Fig. 2 is the molecular structure diagram of malvain-3-O-(6-p-coumaroyl) glucoside;
图3为实施例1中，葡萄皮花色苷粗提液的高效液相色谱图；Fig. 3 is in embodiment 1, the high performance liquid chromatogram of grape skin anthocyanin crude extract;
图4为实施例1中，经大孔树脂分离纯化后，含有芍药素-3-O-(6-对香豆酰)葡萄糖苷、锦葵素-3-O-(6-对香豆酰)葡萄糖苷两个花色苷的洗脱液的高效液相色谱图；Fig. 4 is in Example 1, after separation and purification by macroporous resin, containing paeoniflorin-3-O-(6-p-coumaroyl) glucoside, malvain-3-O-(6-p-coumaroyl) ) the high performance liquid chromatogram of the eluent of two anthocyanins of glucoside;
图5为实施例1中的高速逆流色谱图；Fig. 5 is the high-speed countercurrent chromatogram in embodiment 1;
图6为实施例1中，最终产物芍药素-3-O-(6-对香豆酰)葡萄糖苷单体的高效液相色谱图；Fig. 6 is in embodiment 1, the high performance liquid chromatogram of final product Paeoniflorin-3-O-(6-p-coumaroyl) glucoside monomer;
图7为实施例1中，最终产物锦葵素-3-O-(6-对香豆酰)葡萄糖苷单体的高效液相色谱图；Fig. 7 is in embodiment 1, the high performance liquid chromatogram of final product malvain-3-O-(6-p-coumaroyl) glucoside monomer;
图8为芍药素-3-O-(6-对香豆酰)葡萄糖苷和锦葵素-3-O-(6-对香豆酰)葡萄糖苷的二级质谱图；Fig. 8 is the secondary mass spectrum of paeoniflorin-3-O-(6-p-coumaroyl) glucoside and malvain-3-O-(6-p-coumaroyl) glucoside;
图9为对比例2最终产物的高效液相色谱图。FIG. 9 is a high-performance liquid chromatogram of the final product of Comparative Example 2. FIG.
具体实施方式detailed description
下面结合附图及具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的操作方法，通常按照常规条件，或按照制造厂商所建议的条件。The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The operation method without specifying the specific conditions in the following examples is usually in accordance with the conventional conditions, or in accordance with the conditions suggested by the manufacturer.
实施例1Example 1
将葡萄洗净剥皮，得到1kg葡萄皮，按照料液比1g：5mL的比例加入含0.5％(v/v)盐酸的70％的乙醇溶液充分混合，超声提取60min，(控制温度在50℃以下，避光)，纱布过滤，滤液在4000rpm下离心10min，取上清液。滤渣按相同的方法再提取1次。将滤液合并，使用布氏漏斗再过滤一次。滤液于45℃下减压蒸发除去乙醇并浓缩，得到葡萄皮花色苷粗提液。葡萄皮花色苷粗提液的高效液相色谱图如图3所示。Wash and peel the grapes to obtain 1kg of grape skins, add 70% ethanol solution containing 0.5% (v/v) hydrochloric acid according to the ratio of material to liquid ratio 1g: 5mL, and mix thoroughly, ultrasonically extract for 60min, (control the temperature below 50°C) , protected from light), filtered with gauze, the filtrate was centrifuged at 4000 rpm for 10 min, and the supernatant was taken. The filter residue was extracted once more in the same way. The filtrates were combined and filtered again using a Buchner funnel. The filtrate was evaporated under reduced pressure at 45°C to remove ethanol and concentrated to obtain a crude extract of grape skin anthocyanins. The high-performance liquid chromatogram of the crude extract of grape skin anthocyanins is shown in Figure 3.
将AB-8大孔树脂装入层析柱，并依次使用乙醇，0.5mol/L的盐酸溶 液，0.5mol/L的氢氧化钠溶液，水清洗后，将花色苷粗提液以0.2BV/h的流速注入层析柱中。上样后，依次使用酸水(含0.5％盐酸)，5％、20％、40％、60％的酸性乙醇(含0.5％盐酸)各4倍柱体积洗脱，收集40％和60％酸性乙醇洗脱液，减压蒸发除去乙醇。然后以1：1的比例，使用乙酸乙酯萃取2遍，取水相，适当减压浓缩，冻干后，得到花色苷冻干粉。经大孔树脂分离纯化后，含有芍药素-3-O-(6-对香豆酰)葡萄糖苷、锦葵素-3-O-(6-对香豆酰)葡萄糖苷两个花色苷的洗脱液的高效液相色谱图如图4所示。The AB-8 macroporous resin was loaded into the chromatographic column, and ethanol, 0.5mol/L hydrochloric acid solution, 0.5mol/L sodium hydroxide solution were successively used, and after washing with water, the anthocyanin crude extract was subjected to 0.2BV/L. The flow rate of h is injected into the chromatography column. After loading, use acid water (containing 0.5% hydrochloric acid), 5%, 20%, 40%, and 60% acidic ethanol (containing 0.5% hydrochloric acid) to elute with 4 column volumes each, and collect 40% and 60% acid The ethanol eluent was evaporated under reduced pressure to remove ethanol. Then, in a ratio of 1:1, the mixture was extracted twice with ethyl acetate, and the aqueous phase was taken, concentrated under appropriate reduced pressure, and lyophilized to obtain lyophilized anthocyanin powder. After separation and purification by macroporous resin, it contains two anthocyanins, paeoniflorin-3-O-(6-p-coumaroyl) glucoside and malvain-3-O-(6-p-coumaroyl) glucoside. The high-performance liquid chromatogram of the eluent is shown in Figure 4.
液相分离使用Ultimate XB-C18(7μm,21.2×250mm)制备色谱柱。流动相用纯乙腈(A相)和1.5％的甲酸水溶液(B相)组成。梯度洗脱方法如下：0-4min，5％-20％A相；4-18min，20％-25％A相；18-21min，25％-35％A相；21-24min，35％-60％A相；24-27min，60％-5％A相；27-30min，5％A相，流速为10mL/min，柱温为30℃，检测波长为520nm。将花色苷冻干粉用B相溶解后进样，进样量为4mL，收集22.0-23.5min组分，适当减压浓缩，然后冷冻干燥，得到芍药素-3-O-(6-对香豆酰)葡萄糖苷和锦葵素-3-O-(6-对香豆酰)葡萄糖苷单体粗品。Liquid-phase separation was performed using an Ultimate XB-C18 (7 μm, 21.2×250 mm) preparative chromatographic column. The mobile phase consisted of pure acetonitrile (phase A) and 1.5% formic acid in water (phase B). The gradient elution method is as follows: 0-4min, 5%-20% phase A; 4-18min, 20%-25% phase A; 18-21min, 25%-35% phase A; 21-24min, 35%-60 %A phase; 24-27min, 60%-5%A phase; 27-30min, 5%A phase, flow rate is 10mL/min, column temperature is 30°C, detection wavelength is 520nm. The lyophilized anthocyanin powder was dissolved in phase B and then injected, and the injection volume was 4 mL. The fractions of 22.0-23.5 min were collected, concentrated under appropriate reduced pressure, and then freeze-dried to obtain paeoniflorin-3-O-(6-paraben Crude monomers of myoyl) glucoside and malvain-3-O-(6-p-coumaroyl) glucoside.
乙酸乙酯、水和三氟乙酸按1：1：0.001的体积比置于分液漏斗中，充分摇匀，静置30min后，将上下相分离，分别超声脱气30min。使高速逆流色谱系统的仪器温度稳定在20℃，泵入固定相，之后调节转速至850r/min，正接正转，以2mL/min的流速通入流动相直到平衡，以每3mg冻干粉溶于1mL流动相中的比例溶解花色苷单体粗品冻干粉，使用微孔滤膜过滤后进样，单次进样10mL，并在紫外检测器下检测，检测波长280nm。分别收集90-100min和116-126min的组分(如图5)并减压浓缩，冻干即得到6.8mg的芍药素-3-O-(6-对香豆酰)葡萄糖苷，高效液相色谱图如图6所示，HPLC纯度为99.3％，以及24mg的锦葵素-3-O-(6-对香豆酰)葡萄糖苷，高效液相色谱图如图7所示，HPLC纯度为98.7％。Ethyl acetate, water and trifluoroacetic acid were placed in a separatory funnel in a volume ratio of 1:1:0.001, shaken well, and after standing for 30 min, the upper and lower phases were separated, and ultrasonically degassed for 30 min respectively. Stabilize the instrument temperature of the high-speed countercurrent chromatography system at 20 °C, pump the stationary phase, then adjust the speed to 850 r/min, connect the forward rotation, pass the mobile phase at a flow rate of 2 mL/min until equilibrium, and dissolve the lyophilized powder in every 3 mg. The crude lyophilized powder of anthocyanin monomer was dissolved in 1 mL of mobile phase, filtered with a microporous membrane, and injected into the sample. A single injection of 10 mL was performed and detected under an ultraviolet detector with a detection wavelength of 280 nm. The components of 90-100min and 116-126min were collected respectively (as shown in Figure 5) and concentrated under reduced pressure, and lyophilized to obtain 6.8mg of Paeoniflorin-3-O-(6-p-coumaroyl) glucoside. The chromatogram is shown in Figure 6, the HPLC purity is 99.3%, and 24 mg of malvain-3-O-(6-p-coumaroyl) glucoside, the high performance liquid chromatogram is shown in Figure 7, and the HPLC purity is 98.7%.
将制得花色苷样品在质谱仪中进样，根据质谱图对样品进行分析(图8)，确认分离得到的花色苷质量数正常。The prepared anthocyanin sample was injected into the mass spectrometer, and the sample was analyzed according to the mass spectrum (Fig. 8), and it was confirmed that the mass number of the separated anthocyanin was normal.
实施例2Example 2
将葡萄洗净剥皮，得到2kg葡萄皮，按照料液比1g：6mL的比例加 入含0.5％(v/v)盐酸的80％的乙醇溶液充分混合，超声提取60min，(控制温度在50℃以下，避光)，纱布过滤，滤液在4000rpm下离心10min，取上清液。滤渣按相同的方法再提取1次。将滤液合并，使用布氏漏斗再过滤一次。滤液于45℃下减压蒸发除去乙醇并浓缩，得到葡萄皮花色苷粗提液。The grapes were washed and peeled to obtain 2kg of grape skins, and 80% ethanol solution containing 0.5% (v/v) hydrochloric acid was added according to the ratio of material to liquid ratio of 1g: 6mL to fully mix, and ultrasonically extracted for 60min, (control temperature below 50°C) , protected from light), filtered with gauze, the filtrate was centrifuged at 4000 rpm for 10 min, and the supernatant was taken. The filter residue was extracted once more in the same way. The filtrates were combined and filtered again using a Buchner funnel. The filtrate was evaporated under reduced pressure at 45°C to remove ethanol and concentrated to obtain a crude extract of grape skin anthocyanins.
将AB-8大孔树脂装入层析柱，并依次使用乙醇，0.5mol/L的盐酸溶液，0.5mol/L的氢氧化钠溶液，水清洗后，将花色苷粗提液以0.2BV/h的流速注入层析柱中。上样后，依次使用酸水(含0.5％盐酸)，5％、20％、40％、60％的酸性乙醇(含0.5％盐酸)各4倍柱体积洗脱，收集40％和60％酸性乙醇洗脱液，减压蒸发除去乙醇。然后以1：1的比例，使用乙酸乙酯萃取3遍，取水相，适当减压浓缩，冻干后，得到花色苷冻干粉。The AB-8 macroporous resin was loaded into the chromatographic column, and ethanol, 0.5mol/L hydrochloric acid solution, 0.5mol/L sodium hydroxide solution were successively used, and after washing with water, the anthocyanin crude extract was subjected to 0.2BV/L. The flow rate of h is injected into the chromatography column. After loading, use acid water (containing 0.5% hydrochloric acid), 5%, 20%, 40%, and 60% acidic ethanol (containing 0.5% hydrochloric acid) to elute with 4 column volumes each, and collect 40% and 60% acid The ethanol eluent was evaporated under reduced pressure to remove ethanol. Then, in a ratio of 1:1, the mixture was extracted three times with ethyl acetate, and the aqueous phase was taken, concentrated under appropriate reduced pressure, and freeze-dried to obtain freeze-dried anthocyanin powder.
液相分离使用Ultimate XB-C18(7μm,21.2×250mm)制备色谱柱。流动相用纯乙腈(A相)和1.5％的甲酸水溶液(B相)组成。梯度洗脱方法如下：0-4min，5％-20％A相；4-18min，20％-25％A相；18-21min，25％-35％A相；21-24min，35％-60％A相；24-27min，60％-5％A相；27-30min，5％A相，流速为10mL/min，柱温为30℃，检测波长为520nm。将花色苷冻干粉用B相溶解后进样，进样量为4mL，收集22.0-23.5min组分，适当减压浓缩，然后冷冻干燥，得到芍药素-3-O-(6-对香豆酰)葡萄糖苷和锦葵素-3-O-(6-对香豆酰)葡萄糖苷单体粗品。Liquid-phase separation was performed using an Ultimate XB-C18 (7 μm, 21.2×250 mm) preparative chromatographic column. The mobile phase consisted of pure acetonitrile (phase A) and 1.5% formic acid in water (phase B). The gradient elution method is as follows: 0-4min, 5%-20% phase A; 4-18min, 20%-25% phase A; 18-21min, 25%-35% phase A; 21-24min, 35%-60 %A phase; 24-27min, 60%-5%A phase; 27-30min, 5%A phase, flow rate is 10mL/min, column temperature is 30°C, detection wavelength is 520nm. The lyophilized anthocyanin powder was dissolved in phase B and then injected, and the injection volume was 4 mL. The fractions of 22.0-23.5 min were collected, concentrated under appropriate reduced pressure, and then freeze-dried to obtain paeoniflorin-3-O-(6-paraben Crude monomers of myoyl) glucoside and malvain-3-O-(6-p-coumaroyl) glucoside.
乙酸乙酯、水和三氟乙酸按1：1：0.001的体积比置于分液漏斗中，充分摇匀，静置30min后，将上下相分离，分别超声脱气30min。使高速逆流色谱系统的仪器温度稳定在20℃，泵入固定相，之后调节转速至850r/min，正接正转，以2mL/min的流速通入流动相直到平衡，以每4mg冻干粉溶于1mL流动相中的比例溶解花色苷单体粗品冻干粉，使用微孔滤膜过滤后进样，单次进样10mL，并在紫外检测器下检测，检测波长280nm。分别收集90-100min和116-126min的组分并减压浓缩，冻干即得到12mg的芍药素-3-O-(6-对香豆酰)葡萄糖苷，HPLC纯度为99.3％，以及43mg的锦葵素-3-O-(6-对香豆酰)葡萄糖苷，HPLC纯度为98.4％。Ethyl acetate, water and trifluoroacetic acid were placed in a separatory funnel in a volume ratio of 1:1:0.001, shaken well, and after standing for 30 min, the upper and lower phases were separated, and ultrasonically degassed for 30 min respectively. Stabilize the instrument temperature of the high-speed countercurrent chromatography system at 20 °C, pump the stationary phase, then adjust the speed to 850 r/min, connect the forward rotation, and pass into the mobile phase at a flow rate of 2 mL/min until equilibrium, and dissolve in 4 mg of lyophilized powder. The crude lyophilized powder of anthocyanin monomer was dissolved in 1 mL of mobile phase, filtered with a microporous membrane, and injected into the sample. A single injection of 10 mL was performed and detected under an ultraviolet detector with a detection wavelength of 280 nm. The fractions of 90-100min and 116-126min were collected and concentrated under reduced pressure, and lyophilized to obtain 12 mg of Paeoniflorin-3-O-(6-p-coumaroyl) glucoside, the HPLC purity was 99.3%, and 43 mg of Malvain-3-O-(6-p-coumaroyl)glucoside, HPLC purity 98.4%.
实施例3Example 3
将葡萄洗净剥皮，得到10kg葡萄皮，按照料液比1g：4mL的比例 加入含0.5％(v/v)盐酸的70％的乙醇溶液充分混合，超声提取120min，(控制温度在50℃以下，避光)，纱布过滤，滤液在4000rpm下离心10min，取上清液。滤渣按相同的方法再提取1次。将滤液合并，使用布氏漏斗再过滤一次。滤液于50℃下减压蒸发除去乙醇并浓缩，得到葡萄皮花色苷粗提液。Wash and peel the grapes to obtain 10kg of grape skins, add 70% ethanol solution containing 0.5% (v/v) hydrochloric acid according to the ratio of material to liquid ratio of 1g: 4mL, and mix thoroughly, ultrasonically extract for 120min, (control the temperature below 50°C) , protected from light), filtered with gauze, the filtrate was centrifuged at 4000 rpm for 10 min, and the supernatant was taken. The filter residue was extracted once more in the same way. The filtrates were combined and filtered again using a Buchner funnel. The filtrate was evaporated under reduced pressure at 50°C to remove ethanol and concentrated to obtain a crude extract of grape skin anthocyanins.
将AB-8大孔树脂装入层析柱，并依次使用乙醇，0.5mol/L的盐酸溶液，0.5mol/L的氢氧化钠溶液，水清洗后，将花色苷粗提液以0.2BV/h的流速注入层析柱中。上样后，依次使用酸水(含0.5％盐酸)，5％、20％、40％、60％的酸性乙醇(含0.5％盐酸)各4倍柱体积洗脱，收集40％和60％酸性乙醇洗脱液，减压蒸发除去乙醇。然后以1：1的比例，使用乙酸乙酯萃取2遍，取水相，适当减压浓缩，冻干后，得到花色苷冻干粉。The AB-8 macroporous resin was loaded into the chromatographic column, and ethanol, 0.5mol/L hydrochloric acid solution, 0.5mol/L sodium hydroxide solution were successively used, and after washing with water, the anthocyanin crude extract was subjected to 0.2BV/L. The flow rate of h is injected into the chromatography column. After loading, use acid water (containing 0.5% hydrochloric acid), 5%, 20%, 40%, and 60% acidic ethanol (containing 0.5% hydrochloric acid) to elute with 4 column volumes each, and collect 40% and 60% acid The ethanol eluent was evaporated under reduced pressure to remove ethanol. Then, in a ratio of 1:1, the mixture was extracted twice with ethyl acetate, and the aqueous phase was taken, concentrated under appropriate reduced pressure, and lyophilized to obtain lyophilized anthocyanin powder.
液相分离使用Ultimate XB-C18(7μm,21.2×250mm)制备色谱柱。流动相用纯乙腈(A相)和1.5％的甲酸水溶液(B相)组成。梯度洗脱方法如下：0-4min，5％-20％A相；4-18min，20％-25％A相；18-21min，25％-35％A相；21-24min，35％-60％A相；24-27min，60％-5％A相；27-30min，5％A相，流速为10mL/min，柱温为30℃，检测波长为520nm。将花色苷冻干粉用B相溶解后进样，进样量为4mL，收集22.0-23.5min组分，适当减压浓缩，然后冷冻干燥，得到芍药素-3-O-(6-对香豆酰)葡萄糖苷和锦葵素-3-O-(6-对香豆酰)葡萄糖苷单体粗品。Liquid-phase separation was performed using an Ultimate XB-C18 (7 μm, 21.2×250 mm) preparative chromatographic column. The mobile phase consisted of pure acetonitrile (phase A) and 1.5% formic acid in water (phase B). The gradient elution method is as follows: 0-4min, 5%-20% phase A; 4-18min, 20%-25% phase A; 18-21min, 25%-35% phase A; 21-24min, 35%-60 %A phase; 24-27min, 60%-5%A phase; 27-30min, 5%A phase, flow rate is 10mL/min, column temperature is 30°C, detection wavelength is 520nm. The lyophilized anthocyanin powder was dissolved in phase B and then injected, and the injection volume was 4 mL. The fractions of 22.0-23.5 min were collected, concentrated under appropriate reduced pressure, and then freeze-dried to obtain paeoniflorin-3-O-(6-paraben Crude monomers of myoyl) glucoside and malvain-3-O-(6-p-coumaroyl) glucoside.
乙酸乙酯、水和三氟乙酸按1：1：0.001的体积比置于分液漏斗中，充分摇匀，静置30min后，将上下相分离，分别超声脱气30min。使高速逆流色谱系统的仪器温度稳定在20℃，泵入固定相，之后调节转速至850r/min，正接正转，以2mL/min的流速通入流动相直到平衡。以每5mg冻干粉溶于1mL流动相中的比例溶解花色苷单体粗品冻干粉，使用微孔滤膜过滤后进样，单次进样10mL，并在紫外检测器下检测，检测波长280nm。分别收集90-100min和116-126min的组分并减压浓缩，冻干即得到62mg的芍药素-3-O-(6-对香豆酰)葡萄糖苷，HPLC纯度为98.5％，以及210mg的锦葵素-3-O-(6-对香豆酰)葡萄糖苷，HPLC纯度为98.2％。Ethyl acetate, water and trifluoroacetic acid were placed in a separatory funnel in a volume ratio of 1:1:0.001, shaken well, and after standing for 30 min, the upper and lower phases were separated, and ultrasonically degassed for 30 min respectively. The instrument temperature of the high-speed countercurrent chromatography system was stabilized at 20 °C, the stationary phase was pumped, and then the rotational speed was adjusted to 850 r/min, the positive rotation was connected, and the mobile phase was passed into the mobile phase at a flow rate of 2 mL/min until equilibrium. Dissolve the crude lyophilized powder of anthocyanin monomer in a ratio of 5 mg of lyophilized powder dissolved in 1 mL of mobile phase, filter it with a microporous membrane, inject a sample, inject 10 mL at a time, and detect it under a UV detector. 280nm. The fractions of 90-100min and 116-126min were collected and concentrated under reduced pressure, and lyophilized to obtain 62 mg of Paeoniflorin-3-O-(6-p-coumaroyl) glucoside, the HPLC purity was 98.5%, and 210 mg of Malvain-3-O-(6-p-coumaroyl)glucoside, HPLC purity 98.2%.
对比例1Comparative Example 1
制备工艺与实施例1的相同，区别仅在于改变提取过程不使用酸性乙 醇溶液提取，改用不含酸的乙醇溶液提取。其他步骤不变，则最后目标产物芍药素-3-O-(6-对香豆酰)葡萄糖苷的产率为2mg/kg葡萄皮，远低于6mg/kg葡萄皮；锦葵素-3-O-(6-对香豆酰)葡萄糖苷单体的产率为7mg/kg葡萄皮，远低于20mg/kg葡萄皮。The preparation process is the same as that of Example 1, except that the extraction process is changed not to use acid ethanol solution extraction, but to use acid-free ethanol solution extraction instead. The other steps remain unchanged, the yield of the final target product, paeoniflorin-3-O-(6-p-coumaroyl) glucoside, is 2 mg/kg grape skin, which is much lower than 6 mg/kg grape skin; malvain-3 The yield of -O-(6-p-coumaroyl) glucoside monomer was 7 mg/kg grape skin, which was much lower than 20 mg/kg grape skin.
对比例2Comparative Example 2
制备工艺与实施例1的相同，区别仅在于去掉高速逆流色谱纯化的步骤，其他步骤不变，所得最终产物只能得到芍药素-3-O-(6-对香豆酰)葡萄糖苷与锦葵素-3-O-(6-对香豆酰)葡萄糖苷的混合物，并且该混合物还含有其他杂质(如图9)。The preparation process is the same as that of Example 1, the difference is only that the step of high-speed countercurrent chromatography purification is removed, and other steps remain unchanged, and the final product obtained can only obtain paeoniflorin-3-O-(6-p-coumaroyl) glucoside and bromine. A mixture of quinoline-3-O-(6-p-coumaroyl) glucoside, and this mixture also contains other impurities (Figure 9).
对比例3Comparative Example 3
制备工艺与实施例1的相同，区别仅在于将高速逆流色谱分离的溶剂体系替换为水-正丁醇-甲基叔丁基醚-乙腈-三氟乙酸的体积比5：4：1：2：0.001的体系。经测试，目标化合物芍药素-3-O-(6-对香豆酰)葡萄糖苷与锦葵素-3-O-(6-对香豆酰)葡萄糖苷主要保留在上相中，无法获得目标物。The preparation process is the same as that of Example 1, except that the solvent system separated by high-speed countercurrent chromatography is replaced with water-n-butanol-methyl tert-butyl ether-acetonitrile-trifluoroacetic acid in a volume ratio of 5:4:1:2 : system of 0.001. After testing, the target compounds paeoniflorin-3-O-(6-p-coumaroyl) glucoside and malvain-3-O-(6-p-coumaroyl) glucoside were mainly retained in the upper phase and could not be obtained. Target.
对比例4Comparative Example 4
制备工艺与实施例1的相同，区别仅在于将高速逆流色谱分离的溶剂体系替换为乙酸乙酯-水-三氟乙酸的体积比1:2:0.001的体系。经测试，无法得到目标化合物芍药素-3-O-(6-对香豆酰)葡萄糖苷与锦葵素-3-O-(6-对香豆酰)葡萄糖苷。The preparation process is the same as that of Example 1, except that the solvent system separated by high-speed countercurrent chromatography is replaced with a system with a volume ratio of ethyl acetate-water-trifluoroacetic acid of 1:2:0.001. After testing, the target compounds paeoniflorin-3-O-(6-p-coumaroyl) glucoside and malvain-3-O-(6-p-coumaroyl) glucoside could not be obtained.
对比例5Comparative Example 5
制备工艺与实施例1的相同，区别仅在于将高速逆流色谱分离的溶剂体系替换为乙酸乙酯-水-三氟乙酸的体积比2:1:0.001的体系。经测试，无法得到目标化合物芍药素-3-O-(6-对香豆酰)葡萄糖苷与锦葵素-3-O-(6-对香豆酰)葡萄糖苷。The preparation process is the same as that of Example 1, except that the solvent system separated by high-speed countercurrent chromatography is replaced with a system with a volume ratio of ethyl acetate-water-trifluoroacetic acid of 2:1:0.001. After testing, the target compounds paeoniflorin-3-O-(6-p-coumaroyl) glucoside and malvain-3-O-(6-p-coumaroyl) glucoside could not be obtained.
此外应理解，在阅读了本发明的上述描述内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利 要求书所限定的范围。In addition, it should be understood that after reading the above description of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (7)

  1. 一种分离制备芍药素-3-O-(6-对香豆酰)葡萄糖苷和锦葵素-3-O-(6-对香豆酰)葡萄糖苷的方法，其特征在于，包括步骤：A method for separating and preparing paeoniflorin-3-O-(6-p-coumaroyl) glucoside and malvain-3-O-(6-p-coumaroyl) glucoside, comprising the steps of:
    (1)醇提浓缩：以葡萄为原料，经酸性醇溶液提取，浓缩得到葡萄皮花色苷粗提液；(1) alcohol extraction and concentration: take grape as raw material, extract through acidic alcohol solution, and concentrate to obtain grape skin anthocyanin crude extract;
    (2)大孔树脂纯化：将所述葡萄皮花色苷粗提液注入大孔树脂，经洗脱，浓缩得到花色苷洗脱液；(2) macroporous resin purification: the crude extract of grape skin anthocyanins is injected into macroporous resin, elution, and concentration to obtain anthocyanin eluent;
    (3)萃取：使用有机溶剂萃取所述花色苷洗脱液，再经减压浓缩，冻干后得到花色苷冻干粉；(3) extraction: use organic solvent to extract described anthocyanin eluent, then concentrate under reduced pressure, obtain anthocyanin freeze-dried powder after freeze-drying;
    (4)制备液相色谱纯化：将所述花色苷冻干粉溶解后，注入制备型液相色谱系统中，并用紫外检测器下检测，具体参数条件如下：(4) purification by preparative liquid chromatography: after dissolving the anthocyanin freeze-dried powder, inject it into a preparative liquid chromatography system, and detect with an ultraviolet detector, and the specific parameter conditions are as follows:
    流动相：A相为纯乙腈，B相为甲酸体积百分浓度为1％～2％的甲酸水溶液；Mobile phase: phase A is pure acetonitrile, phase B is formic acid aqueous solution with a volume percent concentration of formic acid of 1% to 2%;
    梯度洗脱程序为：0-4min，5％-20％A相；4-18min，20％-25％A相；18-21min，25％-35％A相；21-24min，35％-60％A相；24-27min，60％-5％A相；27-30min，5％A相；The gradient elution program is: 0-4min, 5%-20% phase A; 4-18min, 20%-25% phase A; 18-21min, 25%-35% phase A; 21-24min, 35%-60 %A phase; 24-27min, 60%-5%A phase; 27-30min, 5%A phase;
    流速为8-10mL/min，柱温为30℃，检测波长为520nm；The flow rate is 8-10mL/min, the column temperature is 30℃, and the detection wavelength is 520nm;
    根据液相色谱图收集保留时间为22.0-23.5min的组分，之后减压蒸发，冻干，得到花色苷单体粗品；The components with retention time of 22.0-23.5min were collected according to the liquid chromatogram, then evaporated under reduced pressure and freeze-dried to obtain the crude anthocyanin monomer;
    (5)高速逆流色谱分离：将乙酸乙酯、水和三氟乙酸以1:1:0.001的体积比混合作为两相溶剂体系，且以上相为固定相，下相为流动相，依次将固定相和流动相泵入高速逆流色谱仪器中，两相在管路中达到平衡后，将花色苷单体粗品用流动相溶解，进样并在紫外检测器下检测，检测波长为280nm，分别收集保留时间为116-126min和90-100min的组分并减压浓缩、冻干，分别得到芍药素-3-O-(6-对香豆酰)葡萄糖苷和锦葵素-3-O-(6-对香豆酰)葡萄糖苷。(5) high-speed countercurrent chromatographic separation: mix ethyl acetate, water and trifluoroacetic acid in a volume ratio of 1:1:0.001 as a two-phase solvent system, and the upper phase is a stationary phase, and the lower phase is a mobile phase, and the stationary phase is sequentially The phase and mobile phase are pumped into the high-speed countercurrent chromatographic instrument. After the two phases reach equilibrium in the pipeline, the crude anthocyanin monomer is dissolved in the mobile phase, and the sample is injected and detected under an ultraviolet detector. The detection wavelength is 280nm, and the collected The components with retention time of 116-126min and 90-100min are concentrated under reduced pressure and freeze-dried to obtain paeoniflorin-3-O-(6-p-coumaroyl)glucoside and malvain-3-O-( 6-p-coumaroyl) glucoside.
  2. 根据权利要求1所述的方法，其特征在于，步骤(1)中，所述酸性醇溶液提取，浓缩得到葡萄皮花色苷粗提液，具体为：将葡萄洗净取皮，与酸性乙醇溶液混合，混匀后打浆，50℃以下超声提取，过滤，滤液在40-50℃减压浓缩除去乙醇，得到葡萄皮花色苷粗提液；The method according to claim 1, characterized in that, in step (1), the acidic alcohol solution is extracted and concentrated to obtain a crude extract of grape skin anthocyanins, specifically: washing the grapes and taking the skins, adding an acidic ethanol solution to the extraction process. Mixing, beating after mixing, ultrasonic extraction below 50°C, filtering, the filtrate is concentrated under reduced pressure at 40-50°C to remove ethanol to obtain a crude extract of grape skin anthocyanins;
    葡萄皮与酸性乙醇溶液的料液比为1g:4-8mL；The material-to-liquid ratio of grape skin and acidic ethanol solution is 1g:4-8mL;
    所述酸性乙醇溶液中，乙醇体积浓度为50％-80％，酸体积浓度为0.1％-1％；In the acidic ethanol solution, the volume concentration of ethanol is 50%-80%, and the volume concentration of acid is 0.1%-1%;
    所述超声提取时间为40-120min。The ultrasonic extraction time is 40-120min.
  3. 根据权利要求2所述的方法，其特征在于，所述酸性乙醇溶液中，酸选自盐酸、甲酸、乙酸、草酸中的至少一种。The method according to claim 2, wherein, in the acidic ethanol solution, the acid is selected from at least one of hydrochloric acid, formic acid, acetic acid, and oxalic acid.
  4. 根据权利要求1所述的方法，其特征在于，步骤(2)中，所述大孔树脂纯化方法具体为：The method according to claim 1, wherein in step (2), the macroporous resin purification method is specifically:
    将葡萄皮花色苷粗提液注入大孔树脂中，之后依次用乙醇体积浓度为0，5％，20％，40％，60％的酸性乙醇溶液各4倍柱体积洗脱，收集乙醇体积浓度为40％和60％的酸性乙醇洗脱液，40-50℃减压蒸发除去乙醇，得到花色苷洗脱液；The crude extract of grape skin anthocyanins was injected into the macroporous resin, and then eluted with 0, 5%, 20%, 40%, and 60% acidic ethanol solutions with 4 times the column volume of each column volume, and the volume concentration of ethanol was collected. 40% and 60% acidic ethanol eluent, evaporated under reduced pressure at 40-50°C to remove ethanol to obtain anthocyanin eluent;
    所述大孔树脂选自AB-8，D101，XAD-7，HPD-100或DM-130，其比表面积为450-550m 2/g，平均孔径为10-50nm，粒径范围在0.3-1.25mm； The macroporous resin is selected from AB-8, D101, XAD-7, HPD-100 or DM-130, its specific surface area is 450-550m 2 /g, the average pore size is 10-50nm, and the particle size is in the range of 0.3-1.25 mm;
    所述酸性乙醇溶液选自酸的体积百分浓度为0.1％～1.5％的乙醇溶液，其中酸选自盐酸、甲酸、乙酸、草酸中的至少一种。The acidic ethanol solution is selected from an ethanol solution with an acid volume percentage concentration of 0.1% to 1.5%, wherein the acid is selected from at least one of hydrochloric acid, formic acid, acetic acid, and oxalic acid.
  5. 根据权利要求1所述的方法，其特征在于，步骤(3)中，所述有机溶剂为乙酸乙酯。The method according to claim 1, wherein in step (3), the organic solvent is ethyl acetate.
  6. 根据权利要求1所述的方法，其特征在于，步骤(4)中，制备型液相色谱系统使用的液相色谱柱为C18柱，单次进样量以花色苷冻干粉计为10-40mg，所述减压蒸发后的体积为蒸发前体积的40％-70％。The method according to claim 1, wherein in step (4), the liquid chromatographic column used in the preparative liquid chromatography system is a C18 column, and the single injection amount is calculated as 10- anthocyanin freeze-dried powder. 40 mg, the volume after evaporation under reduced pressure is 40%-70% of the volume before evaporation.
  7. 根据权利要求1所述的方法，其特征在于，步骤(5)中，高速逆流色谱仪器温度稳定在20-30℃，正接正转，泵入固定相，之后调节转速至800-950r/min，以2mL/min的流速通入流动相并使之平衡，每次进样量以花色苷单体粗品计为20-50mg。The method according to claim 1, characterized in that, in step (5), the temperature of the high-speed countercurrent chromatographic instrument is stabilized at 20-30 ° C, the positive connection is forward rotation, the stationary phase is pumped, and then the rotating speed is adjusted to 800-950 r/min, The mobile phase was passed through and equilibrated at a flow rate of 2 mL/min, and the amount of each injection was 20-50 mg based on the crude anthocyanin monomer.
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